1. Field of the Invention
The invention generally relates to methods of growing filamentous fungi in pellet form in liquid culture. Improved methods for growing filamentous fungi in pellet form in liquid culture include one or more of: 1) addition of a particulate substrate; 2) using spores which have been stored for a period of time prior to inoculation; and 3) using high spore inoculum concentrations, thereby increasing fungal mass and bioproduct formation.
2. Background of the Invention
Filamentous fungal fermentation is widely used to commercially produce useful products such as organic acids, enzymes, antibiotics, and cholesterol lowering drugs (statins) (Cao et al., 1996; Casas Lopez et al., 2004, Chahal, 1985; Hang, 1989; Papagianni, 2004; Schuurmans et al., 1956; Steel et al., 1954). Fungi can be grown in submerged cultures in several different morphological forms: suspended mycelia, clumps, or pellets (Metz et al., 1977). Many studies have discussed the advantages and disadvantages of growth morphologies in terms of different products (Calam, 1976; Konig et al., 1982; Martin et al., 1952).
Without pelletization, filamentous fungi form cotton-like mycelia in liquid culture. This is problematic, because mass transfer of nutrients and products in cotton-like mycelia may be slow due to the large size of mycelial clumps. In bioreactors, clump mycelia increases the viscosity of the medium and wraps around inside elements of the reactor, such as baffles, impellers, heat exchanger, electrode, etc. All of these factors eventually lead to low yield and low productivity. Compared with clump-like mycelia, fungal pellets have a much larger specific surface area which reduces the mass transfer limitations. The pellet morphology also has a beneficial effect on broth rheology, which in return affects momentum, mass and heat transfer in the reactor. Consequently, efficiency of mixing and aeration and cooling systems are enhanced (Charles et al., 1978; Olsvik et al., 1994). It has been reported that higher yields and productivity of lactic acid were obtained using pelletized morphology (Liu et al., 2006a, Yin et al., 1998). Another advantage of fungal pellet fermentation is that the pellets make it possible to perform high biomass concentration cultures to enhance the productivity (Yin et al., 1998; Liu et al., 2006a).
Factors such as medium compositions, pH, medium shear, culture temperature, agitation, additives, oxygen tension, surface-active agents, and medium viscosity have been implicated in pellet formation (Metz et al., 1977; Nielsen et al., 1996; Papagianni, 2004; Znidarsic et al., 1998). Several processes have been able to control the media composition to induce fungal pellet formation. However, pelleting processes described to date have some major disadvantages for industrial applications, e.g. complicated medium compositions which are only suitable for a small range of inoculum, strict reaction conditions, and applicability only to individual strains. For individual strains, each factor has a different importance to the growth morphologies. Some strains such as Rhizopus sp. need strong agitation to form pellets, while some strains such as Penicillium chrysogenum require high pH to form pellets (Metz et al., 1977). Due to the numerous factors affecting pellet formation and growth, presently known methods for culturing fungi in pellet form are thus complex, unpredictable and difficult to adapt to industrial applications.
It has been reported that polymer additives such as anionic polymers of carbopol-934 (carboxypolymethylene) and Reten (polyacrylate) can decrease the agglutination of spores and produce a much more dispersed growth which can increase the pellet number with an accompanying decrease of pellet size and density (Metz, 1977). However, such polymer additives cannot be digested by fungi and become “contaminants” that must be removed prior to or during bioproduct isolation. In addition, some polymer additives are very expensive, while others may be strictly regulated, for example, when used for the production of drugs.
U.S. Pat. No. 6,490,824 (Maekawa et al., 2002) describes the use of water-insoluble growth supporting material to serve as the core of fungus aggregates when culturing basidiomycetous fungus in a liquid culture medium. However, this methodology narrowly targets basidiomycetes, and the patent states that the growth-promoting and stabilizing effect is said to be specific to crushed sugarcane, sugarcane bagasse, wheat bran and pine tree tissues, severely limiting its application.
U.S. Pat. No. 2,850,841 (Szuecs, 1958) describes the liquid culture of edible mushroom mycelia using a support material such as cereal flour (e.g. Cream of Wheat), starch, etc. However, the results of the method were highly variable and inconsistent, ranging from “caviar or pearl-like pellets” to “larger lump- or ball-like masses”. In effect, the method provides little or no control over fungus morphology. Such methodology was geared to the production of edible fungi, would not necessarily apply to other fungi, and would not be suitable for the production of fungus bioproducts in commercial reactors.
The prior art has thus-far failed to provide methods to predictably and consistently grow filamentous fungi in pellet form in liquid culture in a manner that is readily implemented in commercial reactors.